OBJECTIVES: 1. Isolation and characterization of components involved in the mitochondrial electron transport-oxidative phosphorylation system. 2. Study of the enzymatic properties of these components in isolated form and reconstituted systems. 3. Study of the mechanistic details of energy coupling by resolution-reconstitution studies, and by photoaffinity labelling of the mitochondrial coupling sites. 4. Study of structure-function relationships and control mechanisms in mitochondria, sub-mitochondrial particles, electron transfer complexes, and in individual enzyme components of the mitochondrial energy generation-conservation system. To date, this overall strategy has allowed the isolation of 5 enzyme complexes involved in the mitochondrial electron transport and oxidative phosphorylation, and purification of succinate and NADH dehydrogenases. Recent work has established that NADPH can be oxidized by the respiratory chain in complete absence of transhydrogenase activity from NADPH to NAD, and has delineated the site of NADPH interaction with the respiratory chain. Studies on the transhydrogenase and NADPH dehydrogenase enzymes are in progress. Preliminary results suggest that these enzymes can be obtained in soluble form. It has also been possible to obtain preparations of complex V (the ATP-Pi exchange complex) with very high exchange activity (350-470 nmoles.min-1.mg protein) when assayed in presence of added phospholipids. In absence of added lipids, the exchange activity is still considerable (150-195 nmoles.min-1.mg-1 protein). The role of lipids in exchange activity and the possible correlation of exchange activity with vesicle formation are under investigation. Other studies are concerned with the mechanisms of ATP-Pi exchange and energy-linked transhydrogenation. BIBLIOGRAPHIC REFERENCES: Metal-Containing Flavoprotein Dehydrogenases, Hatefi, Y., and Stiggall, D.L. (1976) in The Enzymes (P.D Boyer, ed) ed., vol. 13, Academic Press, New York, pp. 175-297. The Enzymes and Enzyme Complexes of the Mitochondrial Oxidative Phosphorylation System, Hatefi, Y. (1976) in The Enzymes of Biological Membranes, in press.